三疣梭子蟹感染血卵涡鞭虫PCR检测方法的建立

Development of an ITS1 gene-targeted PCR-based diagnosis for Portunus trituberculatus parasite Hematodinium sp.

血卵涡鞭虫病是近年来三疣梭子蟹养殖过程中发生的一种危害较大的流行性疾病，患病蟹的体液和肌肉组织中有大量的血卵涡鞭虫寄生。该病多发生于每年9～11月份的夏秋季节，会在短时间内出现死亡高峰。因该病流行范围广、死亡率高、危害严重，对三疣梭子蟹养殖业发展影响很大。本文根据GenBank中登陆的血卵涡鞭虫18S rDNA和ITS1基因序列设计了1对特异性引物，建立了血卵涡鞭虫病的PCR检测方法，从感染血卵涡鞭虫的三疣梭子蟹组织DNA中扩增出产物大小为585bp的DNA片段。本方法可快速、特异地检测出三疣梭子蟹感染和携带血卵涡鞭虫的状况，为三疣梭子蟹的健康养殖和血卵涡鞭虫病的流行病学调查提供了快速、简便的手段。

The Portunus trituberculatus from Zhoushan is seasonally infected by a parasitic dinoflag-ellate of the genus Hematodinium sp.. Dinoflagellates were found in haemolymph and tissues of infected crabs where the parasite proliferates and causes mortalities. Methods used to detect infection include a morphological index （ pleopod diagnosis） and several immunoassays. The present study describes the development and application of a set of Hematodinium-specific polymerase chain reaction（PCR） primers based on the Hematodinium 18S ribosomal DNA（ 18S rDNA） and the first internal transcribed spacer （ ITS1 ）. In the PCR assay, a diagnostic band 585 bp was consistently amplified from total genomic DNA isolated from Hematodinium-infected Portunus trituberculatus. An effective diagnostic tool is provided for detecting Hematodinium sp. infections and diagnosing epidemic diseases.